4-Aminobutyrate aminotransferase, reaction of P'P2-bis(5'-pyridoxal) diphosphate with lysyl residues connected with catalytic activity.
4-Aminobutyrate aminotransferase is inactivated by preincubation with bispyridoxal-5-P (mixing molar ratio, 20:1) at pH 7.0. The reaction with bispyridoxal-5-P under pseudo-first order conditions proceeds at a slow rate (kobs = 0.03 min-1). The extent of chemical modification was determined by measuring the spectroscopic properties of P-pyridoxyl and P-pyridoxine chromophores formed after reduction of the enzyme reacted with P'P2-bis(5'-pyridoxal) diphosphate. Reduction with NaBH4 results in the incorporation of approximately 2.1 P-pyridoxyl residues/dimer. Thus, the blocking of 2 lysyl residues/dimer is needed for inactivation of the aminotransferase. The time course of inactivation is significantly affected by variations in the pH of the reaction mixtures. Plots of Kobs versus pH indicate the reaction of the bifunctional reagent with lysyl residues characterized by low pK values (pK = 7.3). The substrate alpha-ketoglutarate (10 mM) affords complete protection against inactivation, whereas pyridoxal-5-P failed to prevent the inactivation of the enzyme by bispyridoxal-5-P. It is postulated that binding of alpha-ketoglutarate to lysyl residues is the major factor contributing to stabilization of the catalytic site. Several lines of experimental evidence indicate that inactivation of the aminotransferase cannot be related to dissociation of the cofactor from the catalytic site. The bifunctional reagent bispyridoxal-5-P blocks lysyl residues other than those involved in the binding of the cofactor.
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D007700:Kinetics; D008239:Lysine; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013552:Swine; D000637:Transaminases / Q000378:metabolism*
D001051:Apoenzymes; D011732:Pyridoxal Phosphate; C017076:P(1),P(2)-bis(5'-pyridoxal)diphosphate; D000637:Transaminases; D000612:4-Aminobutyrate Transaminase; D008239:Lysine; C024593:pyridoxine 5-phosphate